DataSheet_1_CD38 marks the exhausted CD8+ tissue-resident memory T cells in hepatocellular carcinoma.docx

IntroductionDespite recent advances in immunotherapy for hepatocellular carcinoma (HCC), the overall modest response rate underscores the need for a better understanding of the tumor microenvironment (TME) of HCC. We have previously shown that CD38 is widely expressed on tumor-infiltrating leukocytes (TILs), predominantly on CD3+ T cells and monocytes. However, its specific role in the HCC TME remains unclear.MethodsIn this current study, we used cytometry time-of-flight (CyTOF), bulk RNA sequencing on sorted T cells, and single-cell RNA (scRNA) sequencing to interrogate expression of CD38 and its correlation with T cell exhaustion in HCC samples. We also employed multiplex immunohistochemistry (mIHC) for validating our findings.ResultsFrom CyTOF analysis, we compared the immune composition of CD38-expressing leukocytes in TILs, non-tumor tissue-infiltrating leukocytes (NIL), and peripheral blood mononuclear cells (PBMC). We identified CD8+ T cells as the dominant CD38-expressing TILs and found that CD38 expression was significantly higher in CD8+ TRM in TILs than in NILs. Furthermore, through transcriptomic analysis on sorted CD8+ TRM from HCC tumors, we observed a higher expression of CD38 along with T cell exhaustion genes, including PDCD1 and CTLA4, compared to the circulating memory CD8 T cells from PBMC. This was validated by scRNA sequencing that revealed co-expression of CD38 with PDCD1, CTLA4, and ITGAE (CD103) in T cells from HCC tumors. The protein co-expression of CD38 and PD-1 on CD8+ T cells was further demonstrated by mIHC on HCC FFPE tissues, marking CD38 as a T cell co-exhaustion marker in HCC. Lastly, the higher proportions of CD38+PD-1+ CD8+ T cells and CD38+PD-1+ TRM were significantly associated with the higher histopathological grades of HCC, indicating its role in the aggressiveness of the disease.ConclusionTaken together, the concurrent expression of CD38 with exhaustion markers on CD8+ TRM underpins its role as a key marker of T cell exhaustion and a potential therapeutic target for restoring cytotoxic T cell function in HCC.

尽管近年来肝细胞癌（HCC）的免疫治疗取得了显著进展，但总体上微弱的反应率凸显了对肝细胞癌肿瘤微环境（TME）的更深入理解的必要性。我们先前已证明CD38在肿瘤浸润性淋巴细胞（TILs）中广泛表达，主要在CD3+ T细胞和单核细胞中。然而，其在HCC TME中的具体作用尚不明确。方法在本项研究中，我们运用了细胞质飞行时间（CyTOF）、排序T细胞的批量RNA测序以及单细胞RNA（scRNA）测序来探究CD38的表达及其与HCC样本中T细胞耗竭的相关性。此外，我们还使用了多重免疫组化（mIHC）来验证我们的发现。结果从CyTOF分析中，我们比较了TILs中表达CD38的淋巴细胞、非肿瘤组织浸润性淋巴细胞（NIL）以及外周血单核细胞（PBMC）的免疫组成。我们确定了CD8+ T细胞为主要的CD38表达TILs，并发现CD38在TILs中的CD8+ TRM表达显著高于NILs。此外，通过对来自HCC肿瘤的排序CD8+ TRM的转录组学分析，我们观察到CD38的表达以及T细胞耗竭基因（包括PDCD1和CTLA4）与来自PBMC的循环记忆CD8 T细胞相比更高。这一发现通过scRNA测序得到了验证，该测序揭示了HCC肿瘤中T细胞CD38与PDCD1、CTLA4和ITGAE（CD103）的共表达。通过HCC FFPE组织上的mIHC进一步证明了CD8+ T细胞上CD38和PD-1的蛋白质共表达，将CD38标记为HCC中T细胞共耗竭的标志物。最后，CD38+PD-1+ CD8+ T细胞和CD38+PD-1+ TRM的高比例与HCC的高组织病理学等级显著相关，表明其在疾病侵袭性中的作用。结论综合来看，CD38与耗竭标志物在CD8+ TRM中的同时表达为其作为T细胞耗竭的关键标志物和恢复HCC中细胞毒性T细胞功能的潜在治疗靶点的角色奠定了基础。